Ldl-cholesterol-lowering cell extract and food supplement

ABSTRACT

An extract of Rhodospirillum rubrum includes rhodovibrin, 1-hydroxyspirilloxanthin, 3,4-didehydrorhodopin, chloroxanthin, bacteriopheophytin a, rhodopin, spirilloxanthin, 3,4-dihydrospirilloxanthin, phytyl derivative of bacteriopheophytin a, ubiquinol-10, ubiquinone-9, ubiquinone-10 and rhodoquinone-10. Such an extract could be used as a medicament, for lowering of LDL-cholesterol in blood plasma of a subject and/or for subjects who suffer from or have an increased risk of one or more of cardiovascular disease, atherosclerosis, dyslipidemia, arteriosclerosis, hypercholesterolemia, familial hypercholesterolemia, hyperlipidemia, an LDL plasma level of at least 70 mg/dL, a total cholesterol level of at least 200 mg/dL, an Lp(a) level of at least 14 mg/dL, inflammation, inflammatory disease, ischemia, or infection.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to a low-density lipoprotein(LDL)-cholesterol-lowering preparation, in particular to a bacterialcell extract for use as a medicament for lowering of the blood plasmaLDL-cholesterol level. More specifically, the present invention relatesto a petroleum ether extract of Rhodospirillum rubrum obtainable byextraction of Rhodospirillum rubrum cells with a mixture of petroleumether with a boiling point of between 60° C. and 80° C. and methanolcomprising sodium chloride. The invention also relates to the petroleumether extract of Rhodospirillum rubrum of the invention, for use in amethod for the lowering of LDL-cholesterol in blood plasma of a subject.The invention further relates to a pharmaceutical preparation, a foodsupplement containing said preparation, a foodstuff containing said foodsupplement, and to a method for their preparation.

BACKGROUND OF THE INVENTION

Cardiovascular disease is caused by a number of synergistic factors, themost important being a too high blood cholesterol level. Cholesterol isan essential building block for animal and human cells, since it is acomponent of cell membranes. Human cells can synthesize their owncholesterol, but cholesterol is also assimilated from food. Bothprocesses play an important part in cholesterol metabolism.

Apart from its essential biological role as a building block forcellular membranes, cholesterol also has negative effects on humanhealth, as a cause of cardiovascular disease (such as, for instance,myocardial infarction, stroke, and peripheral vascular disease), morespecifically in relation to the occurrence of atherosclerotic lesions inthe blood vessel wall. An elevated plasma cholesterol level is the mostimportant predictive risk factor for the occurrence of cardiovasculardisease and atherosclerosis.

In blood plasma, cholesterol is transported in so-called lipoproteins,which can be subdivided into a number of different classes, based ontheir diameter and specific density. The very-low-density lipoproteins(VLDL), the intermediate-density lipoproteins (IDL), the low-densitylipoproteins (LDL), and the high-density lipoproteins (HDL) constitutethe most important classes of lipoproteins.

Experimental and clinical studies have shown that the amount ofcholesterol transported in the VLDL, IDL and LDL classes of lipoproteins(the so-called pro-atherogenic cholesterol) is a risk factor for theoccurrence of cardiovascular disease. Cholesterol transported in HDLparticles, in contrast, protects against the development ofcardiovascular disease (anti-atherogenic cholesterol).

Randomized, placebo-controlled, prospective clinical studies havedemonstrated that lowering plasma cholesterol has a favorable effect onthe incidence of cardiovascular disease and on mortality. A prerequisiteis, though, that the reduction in cholesterol should be predominantly orsubstantially due to a reduction in the pro-atherogenic cholesterolpresent in VLDL, IDL and LDL, leaving the level of anti-atherogeniccholesterol preferably essentially unaltered.

For the treatment and prevention of cardiovascular disease it istherefore imperative to reduce the pro-atherogenic cholesterol, and toincrease, in absolute or relative proportion, the anti-atherogeniccholesterol.

A number of approaches are available to reduce plasma cholesterol. Themost important are:

-   -   to inhibit cholesterol biosynthesis;    -   to increase the removal of cholesterol (and/or its metabolites,        specifically bile acids) from tissues into the intestinal lumen;    -   to reduce the absorption of cholesterol and bile acids from the        gastrointestinal tract.

Drugs that are used to inhibit cholesterol synthesis are ofteninhibitors of the enzyme hydroxymethyl-glutaryl-coenzyme A reductase(HMGCoA reductase), the rate-limiting enzyme in the cholesterolsynthesis pathway. These so-called “statins” are molecules that inhibitenzyme action. Examples are simvastatin, pravastatin and atorvastatin.Statins are generally chemically-synthetized derivatives ofnaturally-occurring fungal metabolites.

To increase cholesterol removal a bile acid-adsorbing resin can be used(for example cholestyramine). Because of the adsorption of bile acids tothe resin, their secretion in the stool is increased, and theirreabsorption from the gut into the blood is reduced, resulting in arelative loss of bile acids from the body. Consequently, the liverincreases the conversion of cholesterol into bile acids, resulting in anet increase in the secretion of cholesterol (metabolites) from thebody. Because bile acids (by solubilizing cholesterol) are essential forthe uptake of cholesterol from the lumen into the intestinal tissue, areduction in bile acid content in the intestinal lumen will also resultin a decreased cholesterol uptake.

Drugs that inhibit the active transport of cholesterol from theintestinal lumen to the blood by inhibiting cellular transport systemsfor cholesterol (and related sterols) in the intestinal epithelial cellsare under development.

Cholesterol Homeostasis

Maintenance of cholesterol homeostasis is vital for healthy status andachieved through a regulatory network consisting of genes involved incholesterol synthesis, absorption, metabolism and elimination. Imbalanceof cholesterol level as a results of environmental and genetic factorsleads to hypercholesterolemia, a predominant risk factor foratherosclerosis (i.e. hardening of furring of the arteries) andassociated coronary and cerebrovascular diseases. Hypercholesterolemiaand its associated cardiovascular diseases represent one of the greatestworldwide economic, social and medical challenges that we are facingnow.

Despite the wide use of therapeutic drugs for controlling bloodcholesterol, like statins inhibiting cholesterol synthesis, the factremains that it is estimated that more than 50% of the population of theUnited States has cholesterol levels at the borderline levels (i.e. >2g/L). In addition, adverse effects associated with therapeutic drugs tocontrol cholesterol levels, such as myopathy, liver damages andpotential drug-drug interaction, have been reported. Therefore,development of additional therapies for controlling cholesterol levelsis warranted, especially for those with better safety profile.

Chinese patent document CN1274584 discloses a preparation ofRhodospirillum for weight-reducing food that reduces blood fat, and amethod for the production of said preparation.

Chinese patent document CN1172653 discloses a liquid Rhodospirillumpreparation for use in the treatment of the human's digestive tract anda method of obtaining said preparation.

Japanese patent document JP08205819 discloses Rhodospirillum culturemedium and Rhodospirillum dried cells for use in a drink or food for usein the treatment of diabetes mellitus, cardiac insufficiency.

None of these documents, however, provides information that a specificpart of the cell and/or a specific fraction of the cell obtained uponsubjecting the cells to a detailed extraction protocol, would beresponsible for the alleged pharmaceutical activity.

Patent EP1569667 discloses a preparation of the membrane fraction ofRhodospirillum for use as a medicament, for use in a food supplement,for use in a foodstuff, wherein said use is for lowering plasmacholesterol. Furthermore, EP1569667 discloses a method for providingsuch membrane fraction of Rhodospirillum, said method comprising thesteps of: culturing a Rhodospirillum cell to a multi-cellular culture;sonicating said cells; separating the membraneous material from thecytoplasmatic material by centrifugation; further washing andrecentrifugation of the pellet retrieved in the previous step.

However, there still exists a need for cholesterol-loweringpreparations, specifically for use in the food industry, and preferablyaimed at human nutrition.

SUMMARY OF THE INVENTION

An aspect of the present invention relates to a petroleum ether extractof Rhodospirillum rubrum by extraction of Rhodospirillum rubrum cellswith a mixture of petroleum ether with a boiling point of between 60° C.and 80° C. and methanol comprising sodium chloride.

An aspect of the invention relates to a petroleum ether extract ofRhodospirillum rubrum obtainable by extraction of Rhodospirillum rubrumcells with a mixture of petroleum ether with a boiling point of between60° C. and 80° C. and methanol comprising sodium chloride, wherein theextract is obtained by extraction for between 10 minutes and 48 hours atbetween 8° C. and 37° C., while mixing the cells with the mixture, andwherein the Rhodospirillum rubrum cells are extracted with the mixturein a volume ratio of between 10:1 and 1:10 of petroleum ether with aboiling point of between 60° C. and 80° C. and methanol comprisingsodium chloride.

Preferred is the petroleum ether extract of Rhodospirillum rubrum of theinvention, wherein the extract is obtained by extraction for between 1hour and 3 hours at a temperature of between 15° C. and 25° C., whilemixing the cells with the mixture, and wherein the Rhodospirillum rubrumcells are extracted with the mixture in a 1:1 volume ratio of petroleumether with a boiling point of between 60° C. and 80° C. and methanolcomprising sodium chloride.

An aspect of the present invention relates to the petroleum etherextract of Rhodospirillum rubrum according to the invention, for use asa medicament.

An aspect of the present invention relates to the petroleum etherextract of Rhodospirillum rubrum according to the invention, for use inthe lowering of LDL-cholesterol in blood plasma of a subject.

An aspect of the present invention relates to the petroleum etherextract of Rhodospirillum rubrum of the invention, for use in a methodfor the lowering of LDL-cholesterol in blood plasma of a subject.

An aspect of the present invention relates to the petroleum etherextract of Rhodospirillum rubrum according to the invention, for use inthe treatment of any of cardiovascular disease, atherosclerosis,dyslipidemia, arteriosclerosis, hypercholesterolemia, familialhypercholesterolemia, hyperlipidemia, an LDL plasma level of at least 70mg/dL, a total cholesterol level of at least 200 mg/dL, an Lp(a) levelof at least 14 mg/dL, inflammation, inflammatory disease, ischemia,infection.

An aspect of the present invention relates to a food supplement withLDL-cholesterol lowering properties, comprising the petroleum etherextract of Rhodospirillum rubrum of the invention.

An aspect of the present invention relates to a foodstuff comprising afood supplement of the invention.

An aspect of the present invention relates to a method for theproduction of a petroleum ether extract of Rhodospirillum rubrum, saidmethod comprising the steps of:

-   -   a) providing Rhodospirillum rubrum cells and providing biphasic        mixture in an about 1:1 volume ratio of petroleum ether with a        boiling point of between 60° C. and 80° C. and methanol        containing about 0.03% by weight sodium chloride;    -   b) mixing the cells of step a) with the mixture of step a)        during between about 1 hour and 3 hours, preferably for about 2        hours at between 18° C. and 24° C.; and    -   c) isolating the petroleum ether extract of Rhodospirillum        rubrum from the methanol containing about 0.03% by weight sodium        chloride, thereby providing the petroleum ether extract of        Rhodospirillum rubrum.

SHORT DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the plasma cholesterol lowering effect with regard toLDL-cholesterol in an in vivo animal model. Animals were fed controlfeed or feed enriched with an extract of Rhodospirillum rubrum.

FIG. 2. Maldi TOF analysis of extracts of Rhodospirillum rubrum.

DETAILED DESCRIPTION OF THE INVENTION

Rhodospirillum is a genus in the family Rhodospirillaceae, a family ofpurple non-sulphur bacteria of the Order Rhodospirillales and the ClassAlpha-proteobacteria. Rhodospirillaceae are, among othercharacteristics, characterized by being phototrophic, and growing bothaerobically and anaerobically, using light as an energy source. To thatpurpose, the bacteria contain chlorophyll b. Within the genusRhodospirillum, several species are distinguished, e.g. Rhodospirillumrubrum, Rhodospirillum centenum, Rhodospirillum photometricum,Rhodospirillum oryzae, Rhodospirillum sulfurexigens, Rhodospirillumsalexigens, Rhodospirillum salinarum, Rhodospirillum sodomense, andRhodospirillum tenue.

Rhodospirillum rubrum is found, among others, in natural waters, in mud,and in sewage treatment plants. The bacterium is used in sewagepurification, for biomass production of animal foodstuff (for example asfeed for poultry and fish), and as a fertilizer. Biomass of phototrophicbacteria is considered an excellent raw material for animal feed becauseof its high content of vitamins and amino acids.

The use of Rhodospirillum rubrum as animal feed has been practiced forsome time. It has been found that R. rubrum cells may importantlycontribute to the prevention of cardiovascular disease by lowering thecholesterol level in blood plasma and/or blood serum (blood).

A cholesterol-lowering property is herein defined as the capability of acomposition, such as a cellular extract, a pharmaceutical composition, apreparation, a food supplement or a foodstuff, when administered to thebody of a subject, such as an animal subject, for example a humansubject, to lower the cholesterol, or LDL-cholesterol, level of theblood of said subject. Methods for measuring the level of cholesterol inblood are known to the skilled person.

A first aspect of the present invention relates to a petroleum etherextract of Rhodospirillum rubrum by extraction of Rhodospirillum rubrumcells with a mixture of petroleum ether with a boiling point of between60° C. and 80° C. and methanol comprising sodium chloride.

A further aspect of the invention relates to a petroleum ether extractof Rhodospirillum rubrum obtainable by extraction of Rhodospirillumrubrum cells with the mixture of petroleum ether with a boiling point ofbetween 60° C. and 80° C. and methanol comprising sodium chloride,wherein the extract is obtained by extraction for between 10 minutes and48 hours at between 8° C. and 37° C., while mixing the cells with themixture, and wherein the Rhodospirillum rubrum cells are extracted withthe mixture in a volume ratio of between 10:1 and 1:10 of petroleumether with a boiling point of between 60° C. and 80° C. and methanolcomprising sodium chloride.

Throughout the specification the term “petroleum ether with a boilingpoint of between 60° C. and 80° C.” has its regular scientific meaningand here refers to a petroleum fraction consisting of aliphatichydrocarbons and boiling in the range of between 60° C. and 80° C.

A preparation of Rhodospirillum spp. is herein defined as an amount ofcellular material of Rhodospirillum spp. which has been processed insome way. A preparation of Rhodospirillum spp. is, according to thepresent invention, a petroleum ether extract with cholesterol-loweringproperties, such as the petroleum ether extract of Rhodospirillum rubrumby extraction of Rhodospirillum rubrum cells with a mixture of petroleumether with a boiling point of between 60° C. and 80° C. and methanolcomprising sodium chloride, according to the invention.

A preparation according to the invention consists of a petroleum etherextract derived from one species from the genus Rhodospirillum, or apreparation according to the invention consists of a petroleum etherextract derived from mixtures of different Rhodospirillum spp. selectedfor example from Rhodospirillum rubrum, Rhodospirillum centenum,Rhodospirillum photometricum, Rhodospirillum oryzae, Rhodospirillumsulfurexigens, Rhodospirillum salexigens, Rhodospirillum salinarum,Rhodospirillum sodomense, and Rhodospirillum tenue.

The genus Phaeospirillum is a different member of the family ofRhodospirillaceae, which genus comprises Phaeospirillum fulvum,Phaeospirillum chandramohanii, Phaeospirillum oryzae, Phaeospirillumtilakii and Phaeospirillum molischianum. Therefore, alternatively apreparation according to the invention consists of a petroleum etherextract derived from one species from the genus Phaeospirillum, or apreparation according to the invention consists of a petroleum etherextract derived from mixtures of different

Phaeospirillum spp. selected for example from Phaeospirillum fulvum,Phaeospirillum chandramohanii, Phaeospirillum oryzae, Phaeospirillumtilakii and Phaeospirillum molischianum.

Of course, a further alternative preparation according to the inventionconsists of a petroleum ether extract derived from at least one speciesfrom the genus Phaeospirillum and at least one species from the genusRhodospirillum.

Preferably, a preparation of Rhodospirillum spp. and/or ofPhaeospirillum spp. comprises a petroleum ether extract ofRhodospirillum rubrum and/or of Phaeospirillum molischianum, still morepreferably of the species Rhodospirillum rubrum strain ATCC 11170(strain DSM 467) or strain ATCC 25903 and/or of the Phaeospirillummolischianum strain DSM 120 (ATCC, American Type Culture Collection;DSMZ, Deutsche Sammlung von Mikroorganismen and Zellkulturen).

A preparation of Rhodospirillum spp. and/or of Phaeospirillum spp.according to the invention, may contain 20-100% (w/w), preferably40-100% (w/w), even more preferably 60-100% (w/w), and optimally 80-100%(w/w) of cellular material from Rhodospirillum spp. and/or fromPhaeospirillum spp., said cellular material being the petroleum etherextract of cells according to the invention. In addition, a preparationmay contain other components, depending upon the way the selectedpreparation is to be prepared. For instance, a preparation may stillcontain water, or, in the case of a freeze-dried preparation, forexample glycerol or sucrose or both.

In a preferred embodiment, a freeze-dried preparation of Rhodospirillumspp. and/or of Phaeospirillum spp. comprising the petroleum etherextract according to the invention is mixed with filling materials suchas microcrystalline cellulose (MCC) or mannitol, with a binder such ashydroxypropyl-cellulose (HPC), and/or lubricants, such as stearic acidand/or other excipients, and pelleted as a dry powder, or prepared forapplication in a different way.

The petroleum ether extract of Rhodospirillum rubrum of the invention ispreferably a petroleum ether extract that is obtained by extraction forbetween 1 hour and 3 hours, preferably for about 2 hours, at between 15°C. and 25° C., preferably at about 18° C. and 24° C., more preferably atabout room temperature while mixing the cells with the mixture ofpetroleum ether with a boiling point of between 60° C. and 80° C. andmethanol comprising sodium chloride.

Preferred is the petroleum ether extract of Rhodospirillum rubrumaccording to the invention, wherein the extract is obtained byextraction for between 20 minutes and 24 hours, preferably between 30minutes and 16 hours, more preferably between 45 minutes and 8 hours,most preferably between 1 hour and 3 hours, at a temperature of between10° C. and 30° C., preferably between 12° C. and 27° C., more preferablybetween 15° C. and 25° C., while mixing the cells with the mixture.

In an embodiment of the invention, the petroleum ether extract ofRhodospirillum rubrum of the invention is by extraction for about 2hours at room temperature while mixing the cells with the mixture ofpetroleum ether with a boiling point of between 60° C. and 80° C. andmethanol comprising sodium chloride.

The term “room temperature” as used herein has its conventional meaningand here refers to a temperature of between 20° C. and 22° C.

The petroleum ether extract of Rhodospirillum rubrum of the invention ispreferably upon centrifugation for between about ten minutes and 60minutes, preferably for about 20 minutes, at between 18° C. and 24° C.,preferably at about 20° C. and 22° C., more preferably at roomtemperature of the mixture of petroleum ether with a boiling point ofbetween 60° C. and 80° C. and methanol comprising sodium chloride afterincubation of Rhodospirillum rubrum cells with said mixture.

Preferred is the petroleum ether extract of Rhodospirillum rubrumaccording to the invention, wherein the Rhodospirillum rubrum cells areextracted with the mixture of petroleum ether with a boiling point ofbetween 60° C. and 80° C. and methanol containing sodium chloride, in avolume ratio of between 8:1 and 1:8, preferably between 5:1 and 1:5,more preferably between 2:1 and 1:2, most preferably in a 1:1 volumeratio of petroleum ether with a boiling point of between 60° C. and 80°C. and methanol comprising sodium chloride.

Thus, the petroleum ether extract of Rhodospirillum rubrum of theinvention is preferably by extracting the Rhodospirillum rubrum cellswith a mixture in an about 1:1 volume ratio, preferably a 1:1 volumeratio of petroleum ether with a boiling point of between 60° C. and 80°C. and methanol containing sodium chloride.

Particularly preferred is the petroleum ether extract of Rhodospirillumrubrum of the invention, wherein the extract is obtained by extractionfor between 1 hour and 3 hours at a temperature of between 15° C. and25° C., while mixing the cells with the mixture, and wherein theRhodospirillum rubrum cells are extracted with the mixture in a 1:1volume ratio of petroleum ether with a boiling point of between 60° C.and 80° C. and methanol comprising sodium chloride.

The petroleum ether extract of Rhodospirillum rubrum of the invention ispreferably obtained by extracting the Rhodospirillum rubrum cells with amixture of petroleum ether with a boiling point of between 60° C. and80° C. and methanol comprising about between 0.02% and 0.04% by weightsodium chloride, preferably comprising about 0.03% by weight sodiumchloride.

The petroleum ether extract of Rhodospirillum rubrum of the inventionpreferably comprises one or more carotenoids and/or one or morequinones. Preferably, the petroleum ether extract of Rhodospirillumrubrum of the invention comprises one or more carotenoids and one ormore quinones.

The petroleum ether extract of Rhodospirillum rubrum of the inventionpreferably comprises one or more carotenoids selected from thecarotenoids rhodovibrin, 1-hydroxy-spirilloxanthin,3,4-didehydrorhodopin, chloroxanthin, bacteriopheophytin a, rhodopin,spirilloxanthin, 3,4-dihydrospirilloxanthin and phytyl derivative ofbacteriopheophytin a, and/or one or more quinones selected from thequinones ubiquinol-10, ubiquinone-9, ubiquinone-10 and rhodoquinone-10.

The petroleum ether extract of Rhodospirillum rubrum of the inventionpreferably comprises rhodovibrin, 1-hydroxy-spirilloxanthin,3,4-didehydrorhodopin, chloroxanthin, bacteriopheophytin a, rhodopin,spirilloxanthin, 3,4-dihydrospirilloxanthin, phytyl derivative ofbacteriopheophytin a, ubiquinol-10, ubiquinone-9, ubiquinone-10 andrhodoquinone-10.

Without wishing to be bound by theory, Parson & Rudney, in The Journalof Biological Chemistry (Vol. 240, No. 4, April 1965), report on thebiosynthesis of ubiquinone and rhodoquinone from p-hydroxybenzoate andn-hydroxybenzaldehyde in Rhodospirillum rubrum. Data are presented bythese authors which indicate that growing R. rubrum catabolizesubiquinone to the derivative rhodoquinone.

Preferably, the petroleum ether extract of Rhodospirillum rubrum of theinvention comprises compounds with a molecular weight of at least 2100Da giving rise to a peak having an m/z value of 752.7 and/or of 769.3 ina nano-electron spray ionization-mass spectrometry (nanoESI-MS) spectrumobtained with, for example, an IonTrap nanoESI-MS high-resolution tripletime-of-flight mass spectrometer (Bruker), said one peak or said twopeaks with an m/z value of 752.7 and/or of 769.3 having an intensity inthe nanoESI-MS spectrum that is at least twice the intensity of at leastone, preferably of both of the further peaks in the nanoESI-MS spectrumhaving an m/z value of 665.8 and 883.6, preferably about five times theintensity, according to the invention.

Preferably, the petroleum ether extract of Rhodospirillum rubrum of theinvention comprises compounds with a molecular weight of at least 2100Da giving rise to peaks having an m/z value of 752.7 and 769.3 in ananoESI-MS spectrum obtained with for example an IonTrap nanoESI-MShigh-resolution triple time-of-flight mass spectrometer (Bruker), saidpeaks with an m/z value of 752.7 and 769.3 having an intensity in thenanoESI-MS spectrum that is at least twice the intensity of furtherpeaks in the nanoESI-MS spectrum having an m/z value of 665.8 and 883.6,preferably about five times the intensity, according to the invention.

Preferably, the nanoESI-MS analysis and measurement with the petroleumether extract of Rhodospirillum rubrum of the invention is performedwith said petroleum ether extract diluted in 1% v/v formic acid inacetonitrile, according to the invention.

Preferably, the petroleum ether extract of Rhodospirillum rubrum of theinvention comprises compounds giving rise to one or more peaks,preferably three peaks, of the peaks having an m/z value of 500.3, 882.5and 898.5 in a matrix-assisted laser desorption ionizationtime-of-flight (MALDI-ToF) mass spectrometry (MS) spectrum, said one ormore peaks with an m/z value of 500.3, 882.5 and 898.5 respectively,having an intensity in the MALDI-ToF MS mass spectra in the ratio ofabout 1:1:2,

preferably about 1:1:3,

more preferably about 1:1:4, according to the invention.

Preferably, the petroleum ether extract of Rhodospirillum rubrum of theinvention comprises compounds giving rise to at least three peaks, saidpeaks having an m/z value of 500.3, 882.5 and 898.5 respectively in aMALDI-ToF MS spectrum, said three peaks with an m/z value of 500.3,882.5 and 898.5 respectively, having an intensity in the MALDI-ToF MSmass spectra in the ratio of about 1:1:2, preferably about 1:1:3, morepreferably about 1:1:4, according to the invention.

The aforementioned MALDI-ToF MS mass spectra are for example obtainedwith a Qtof Premier mass spectrometer (Waters) equipped with a Nd:YAGlaser, operating at 355 nm with an output frequency of 50 Hz whereintime-of-flight mass analyses are performed in reflection mode at aresolution of about 10.000. Then, the petroleum ether extract of theinvention is preferably analyzed using(DCTB)trans-2-[3-(4-tertbutylphenyl)-2-methylprop-2-enylidene]malononitrile matrix.

Preferably, the aforementioned MALDI-ToF MS analysis and measurementwith the petroleum ether extract of Rhodospirillum rubrum of theinvention are performed with said petroleum ether extract dissolved intetrahydrofuran, according to the invention.

Preferably, the petroleum ether extract of Rhodospirillum rubrum of theinvention comprises compounds giving rise to at least two peaks, said atleast two peaks having an m/z value of 651.6 and 647.6 respectively, ina nanoESI-MS spectrum obtained with for example a triple time-of-flightmass spectrometer (ABSCIEX), said two peaks with an m/z value of 651.6and 647.6 respectively having an intensity in the nanoESI-MS spectrumwith a ratio of about 1:1, preferably about 5:4, more preferably about2:1, according to the invention.

Preferably, the petroleum ether extract of Rhodospirillum rubrum of theinvention comprises compounds giving rise to at least two peaks, said atleast two peaks having an m/z value of 881.5 and 927.5 respectively, ina nanoESI-MS spectrum obtainable with for example a tripletime-of-flight mass spectrometer (ABSCIEX), said two peaks with an m/zvalue of 881.5 and 927.5 respectively having an intensity in thenanoESI-MS spectrum with a ratio of about 1:1, preferably about 3:2,more preferably about 2:1, according to the invention.

Preferably, the aforementioned nanoESI-MS analysis and measurement withthe petroleum ether extract of Rhodospirillum rubrum of the inventionusing an ABSCIEX apparatus are performed with said petroleum etherextract dissolved in a solution consisting of 99% v/v acetonitrile and1% v/v formic acid, according to the invention.

A further aspect of the present invention relates to the petroleum etherextract of Rhodospirillum rubrum according to the invention, for use asa medicament.

An aspect of the present invention relates to the petroleum etherextract of Rhodospirillum rubrum according to the invention, for use inthe lowering of LDL-cholesterol in blood plasma of a subject.

An aspect of the invention relates to the petroleum ether extract ofRhodospirillum rubrum according to the invention, for use in a methodfor the lowering of LDL-cholesterol in blood plasma of a subject.Similarly, an aspect of the invention relates to the use of thepetroleum ether extract of Rhodospirillum rubrum according to theinvention for the manufacture of a medicament for the treatment of highLDL-cholesterol in blood plasma of a subject. Furthermore, an aspect ofthe invention relates to the use of the petroleum ether extract ofRhodospirillum rubrum according to the invention for the manufacture ofa medicament for the lowering of LDL-cholesterol in blood plasma of asubject.

An aspect of the present invention relates to the petroleum etherextract of Rhodospirillum rubrum according to the invention, for use inthe treatment of any of cardiovascular disease, atherosclerosis,dyslipidemia, arteriosclerosis, hypercholesterolemia, familialhypercholesterolemia, hyperlipidemia, an LDL plasma level of at least 70mg/dL, a total cholesterol level of at least 200 mg/dL, an Lp(a) levelof at least 14 mg/dL, inflammation, inflammatory disease, ischemia,infection.

In an embodiment of the invention, the use of the petroleum etherextract of Rhodospirillum rubrum of the invention in the treatment ofany of cardiovascular disease, atherosclerosis, dyslipidemia,arteriosclerosis, hypercholesterolemia, familial hypercholesterolemia,hyperlipidemia, an LDL plasma level of at least 70 mg/dL, a totalcholesterol level of at least 200 mg/dL, an Lp(a) level of at least 14mg/dL, inflammation, inflammatory disease, ischemia, infection, is forlowering of LDL-cholesterol in blood plasma of a subject suffering fromor having an increased for any of said aforementioned diseases or healthissues.

The aforementioned preparations of Rhodospirillum spp. and/orpreparations of Phaeospirillum spp. are very suitable petroleum etherextracts for use as a medicament or as a pharmaceutical preparation tolower plasma cholesterol levels, preferably in human plasma.

Preferably, a petroleum ether extract of Rhodospirillum rubrum accordingto the invention is for use as a medicament according to the invention,wherein said use is in the lowering of LDL-cholesterol in blood plasmaof a subject.

In an embodiment of the invention, the petroleum ether extract ofRhodospirillum rubrum is used for the lowering of LDL-cholesterol inblood plasma of a subject, wherein the subject suffers from or has anincreased risk for any one or more of cardiovascular disease,atherosclerosis, dyslipidemia, arteriosclerosis, hypercholesterolemia,familial hypercholesterolemia, hyperlipidemia, an LDL plasma level of atleast 70 mg/dL, a total cholesterol level of at least 200 mg/dL, anLp(a) level of at least 14 mg/dL, inflammation, inflammatory disease,ischemia, infection.

The petroleum ether extract of Rhodospirillum rubrum of the inventionpreferably lowers the level of LDL-cholesterol in blood plasma, whereasthe level of total cholesterol is essentially unaltered and/or whereasthe level of HDL-cholesterol is essentially unaltered, when thepetroleum ether extract of Rhodospirillum rubrum of the invention is foruse in the lowering of LDL-cholesterol in blood plasma of a subject.

Surprisingly, the inventors found that the petroleum ether extract ofRhodospirillum rubrum of the invention for use as a medicament or as apharmaceutical preparation to lower plasma cholesterol levels, lowersLDL-cholesterol in plasma with at least 40%, e.g. for about 46%, whereasat the same time both the level of total cholesterol and the level ofHDL-cholesterol are essentially unaltered.

Preferably, the petroleum ether extract of Rhodospirillum rubrum of theinvention for use as a medicament or as a pharmaceutical preparation tolower plasma cholesterol levels, lowers LDL-cholesterol in plasma withat least 20%, preferably with about 40 to 80%, more preferably withabout 50%, whereas at the same time both the level of total cholesteroland the level of HDL-cholesterol are essentially unaltered, according tothe invention.

The petroleum ether extract of Rhodospirillum rubrum according theinvention, for use in the lowering of LDL-cholesterol in blood plasma ofa subject according to the invention, is preferably administered orally.

Various embodiments of a preparation comprising a petroleum etherextract of Rhodospirillum spp. and/or of Phaeospirillum spp. accordingto the present invention are equally possible. For example, apreparation of the invention can be supplied as a fluid preparation ofthe invention containing solid components suspended, dispersed oremulsified in an aqueous solution, providing a composition according tothe invention. Such a composition of the invention can be used directlyas a preparation comprising a petroleum ether extract of Rhodospirillumspp. and/or of Phaeospirillum spp. according to the invention, or can beprocessed into a food supplement in an alternative embodiment.

An aspect of the current invention relates to a food supplement withLDL-cholesterol lowering properties, comprising the petroleum etherextract of Rhodospirillum rubrum according to the invention.

In the present invention a food supplement is defined as a formulationthat is consumed in addition to a normal diet and that containscomponents that do not occur in a normal diet, or that occur in lowamounts or in insufficient amounts, while sufficient or increasedconsumption of these components is desired. Preferably, a foodsupplement according to the invention is composed such that it issuitable for human consumption. Consequently, a food supplement asdefined in the present invention should preferably have a texture, tasteand smell, but also a nutritional value, that makes the supplementsuitable for human consumption.

In embodiments of the present invention a food supplement withcholesterol-lowering properties comprises a preparation ofRhodospirillum spp. and/or of Phaeospirillum spp., said preparationcomprising a petroleum ether extract of Rhodospirillum spp. and/or ofPhaeospirillum spp. according to the present invention.

A food supplement according to the invention preferably contains between0.1% and 99.9% (w/w) of a preparation of Rhodospirillum spp. comprisinga petroleum ether extract of Rhodospirillum spp. Preferably, a foodsupplement contains between 10% and 90% (w/w), even more preferablybetween 30% and 75% (w/w), of a preparation of Rhodospirillum spp and/orof Phaeospirillum spp, according to the invention.

To make a food supplement comprising a preparation of Rhodospirillumspp. and/or of Phaeospirillum spp. comprising a petroleum ether extractof Rhodospirillum spp. and/or of Phaeospirillum spp. suitable forconsumption, components are preferably added to improve, for instance,texture, taste or smell, according to the invention.

Consequently, a food supplement according to the invention preferablycomprises (additional) sources of protein, carbohydrate and fat, andvitamins, minerals, electrolytes, trace elements, and other suitablecomponents, so that the food supplement itself is suitable for use as anourishing food.

As a source of protein each and every protein that is suitable for usein nutritional formulations, and mixtures of these, are preferably usedin a food supplement according to the invention. This type of proteinsencompasses for instance animal proteins such as whey proteins, wheyprotein concentrates, whey powder, egg protein, egg albumin, casein, ormilk albumin, and plant proteins such as soy protein, soy meal, orproteins from soy milk. For choosing the source of proteins to be used,the biological value of a protein may constitute an important criterion.Caseinate, including calcium caseinate, but also whey, milk albumin, eggalbumin, and total egg proteins, for instance, are proteins with a veryhigh biological value, because they contain a large proportion ofessential amino acids.

Suitable carbohydrates to be used in a food supplement according to theinvention are, for instance, preferably simple short-chain carbohydratessuch as mono- and disaccharides, but also polysaccharides, or acombination of both. A carbohydrate is preferably selected because ofits suitable organoleptic properties, according to the invention.Preferably, a complex carbohydrate is suitably used as a food fiber,according to the invention.

A food supplement according to the invention preferably contains, insome embodiments, combinations of both simple and complex carbohydrates.A food supplement according to the invention preferably contains, insome embodiments, a fat selected from all edible oils and edible fats.

Vitamins and minerals are preferably added to a preparation according tothe invention, in conformity with the rules of the regulatory healthauthorities, and preferably encompasses all vitamins and mineralsendorsed by the above authorities, for instance vitamin A, B1, B2, B12,C, D, B, and K, and folic acid, niacin, pantothenic acid, and biotin. Asminerals for instance iron, zinc, iodine, calcium, magnesium, chromium,and selenium are preferably added to a preparation according to theinvention.

Electrolytes such as the ions of sodium, potassium, and chloride, andtrace elements and other additives do preferably also form part of afood supplement according to the invention. Such components are, ifpresent, preferably used in the recommended concentrations.Additionally, a food supplement according to the invention preferablycontains components improving its texture, colorings and flavorings,aromatic substances, spices, fillers, emulsifiers, stabilizingcompounds, preservatives, antioxidants, fibers, and other supplementssuch as amino acids, choline, lecithin, fatty acids, etc. The choice ofsuch components depends upon formulation, design, and preferences. Theamounts of such components that are added are known to the skilledperson, while the choice of the amounts to be added are preferablyguided by considering the recommended daily amounts (RDA) for childrenand adults.

Emulsifiers are preferably added to stabilize the final product of theinvention. Examples of acceptable emulsifiers are lecithin (e.g.,derived from soy or from egg), and/or mono- and di-glycerides, accordingto the invention. As stabilizers, carob, guar or carrageenan are, forinstance, preferably used, according to the invention.

Preservatives are preferably added to increase the shelf life of theproduct of the invention.

Preferably, preservatives such as sodium sorbate, potassium sorbate,potassium benzoate, sodium benzoate, or calcium disodium EDTA are usedin a preparation of the invention.

In addition to the carbohydrates mentioned above, natural or syntheticsweeteners, such as saccharides, cyclamates, aspartame, acesulfamepotassium, and/or sorbitol, are preferably added to the food supplement,according to the invention.

The amounts of food supplement of the invention to be consumed arevarying in size, and are not necessarily restricted to the dosagesmentioned in the dosages advised. The term “food supplement” is notmeant to be restricted to a specified weight, or to a specified dose ofthe food supplement.

The composition of a food supplement according to the invention takes inprinciple any form that is suitable for human or animal consumption,according to the invention.

In a preferred embodiment of the invention, the food supplement is a drypowder that is suitable to be suspended, dispersed or emulsified in anaqueous solution such as coffee, tea, broth, and fruit juice. To thatend, the powder is preferably supplied in a dispenser according to theinvention.

In an alternative preferred embodiment of the invention, the foodsupplement is formulated, starting from dry powder, as a tablet. To thisend, preferably the composition of a food supplement according to theinvention is suitably supplied with fillers such as microcrystallinecellulose (MCC) and mannitol, binders such as hydroxylpropyl-cellulose(HPC), lubricants such as stearic acid, and other excipients.

A food supplement according to the invention is in one embodimentpreferably supplied as a fluid, in which the solid components have beensuspended, dispersed or emulsified. Such a composition of the inventionis preferably directly mixed into a foodstuff, or is preferably forinstance extruded and formatted into granules or other forms.

In an alternative embodiment of the invention, a food supplement ispreferably formulated in a solid form, such as a bar, a biscuit, or aroll.

A food supplement of the invention is preferably formulated for oralconsumption, preferably in combination with an acceptable carrier suchas a capsule, a tablet, a water-miscible powder, or another formacceptable for administration. Alternatively, a food supplement of theinvention is preferably processed into a foodstuff, according to theinvention.

One aspect of the present invention relates to a foodstuff comprising afood supplement according to the invention.

Other aspects of the present invention relate to ways of producing apreparation, a food supplement, or a foodstuff according to theinvention.

A further aspect of the present invention relates to a method for theproduction of a petroleum ether extract of Rhodospirillum rubrum of theinvention, said method comprising the steps of:

(a) providing Rhodospirillum rubrum cells and providing biphasic mixturein a 1:1 volume ratio of petroleum ether with a boiling point of between60° C. and 80° C. and methanol containing about 0.03% by weight sodiumchloride;

(b) mixing the cells of step a) with the mixture of step a) duringbetween about 1 hour and 3 hours, preferably for about 2 hours atbetween 18° C. and 24° C., preferably at about 20° C. and 22° C., morepreferably at room temperature; and

(c) isolating the petroleum ether extract of Rhodospirillum rubrum fromthe methanol containing about 0.03% by weight sodium chloride, therebyproviding the petroleum ether extract of Rhodospirillum rubrum.

A method for the production of a preparation according to the inventionpreferably involves the steps necessary for culturing cells of one ormore Rhodospirillum spp. and/or Phaeospirillum spp., to harvest thecells in the said culture, and to process the cells of the said cultureinto a preparation.

Details of such methods are, among others, described in the examplesmentioned below. The skilled person will understand that variousalternative methods can be used.

During the culturing of cells of Rhodospirillum spp. and/or ofPhaeospirillum spp. anaerobic and phototrophic conditions are applied.As a carbon source, various organic nutrients are preferably used. Verysuitable culture media and growth conditions for cells of Rhodospirillumspp. and/or of Phaeospirillum spp. are for example “Segers andVerstraete medium” (Segers and. Verstraete, 1983), using lactic acid(about 2.7 gram/L) as a carbon source, at a pH of about 6.8-6.9, and ata temperature of 25-37° C., preferably adapted to the specificrequirements of the micro-organism involved, at constant light intensityfrom for instance strip lighting (light intensity 300 μM quanta·m⁻²·s⁻¹)and anaerobically. Other media suitable for culturing Rhodospirillumspp. and/or for culturing Phaeospirillum spp. are for example “modifiedRhodospirillaceae medium” (DSMZ medium #27, DMSZ GmbH, Braunschweig,Germany), or Cens medium (DSMZ medium #748). The cells are suitable tobe cultured to a density of between 0.01 mg/mL and 50 mg/mL, preferablybetween 1 mg/mL and 5 mg/mL based on the wet weight of the cells.

Cells are equally grown anaerobically at 30° C. in 1 liter flaskscontaining a medium constituting 3.1 ml/L 60% DL-lactate solution, 3 g/Lbacteriological peptone and 3 g/L yeast extract in tap water, the pH ofthe medium being 6.8, and illuminated with an average photon radiationstrength of 50 μM quanta·m⁻²·s⁻¹, using 3 Tungsten lamps of 40 W. After3 days of growth, the optical density at 660 nm amounted 3.5 (1.2 g/kgdry weight).

Once the cells have reached a suitable cell density, they are processedinto a preparation according to the invention using separation from thegrowth medium or harvesting by, for instance, centrifugation orfiltration.

The concentrated cell mass is then used, after further processing, as apreparation according to the invention.

The further steps in the processing of cell material of Rhodospirillumspp. and/or of Phaeospirillum spp. to obtain a usable preparationcomprising the petroleum ether extract of Rhodospirillum spp. and/or ofPhaeospirillum spp. of the invention, involves, for example, a washingstep, and also involves further processing of the cells by extraction,and optionally freeze-drying.

Minnikin, O'Donnell et al. have described an integrated procedure forthe extraction of bacterial isoprenoid quinones and polar lipids(Journal of Microbiological Methods 2 (1984) pp. 233-241). A procedurefor the sequential extraction of isoprenoid quinones and polar lipidsfrom bacterial cells is outlined by these authors. Extraction with abiphasic mixture of petroleum ether (b.p. 60-80° C.) and methanolicsaline gave an upper phase containing isoprenoid quinones. As examplesof the procedure, the isoprenoid quinones of Bacillus subtilis,Mycobacterium avium, Pseudomonas diminuta and Streptomyces griseus wereextracted and analyzed.

An aspect of the invention relates to the petroleum ether extract ofRhodospirillum rubrum obtainable by the method of the invention.

A food supplement according to the invention may suitably be used toreduce intestinal cholesterol absorption, thus reducing the cholesterollevel of blood plasma.

In another embodiment of the invention, a food supplement of theinvention is applied in a foodstuff with cholesterol-loweringproperties.

A method to prepare a cholesterol-lowering foodstuff of the inventioninvolves the production of a foodstuff incorporating a food supplementaccording to the invention. Such a method preferably involves a step inwhich a foodstuff is first prepared in the normal way, followed by theaddition of a preparation of Rhodospirillum spp. and/or ofPhaeospirillwn spp. to the prepared foodstuff. Also, it is possible toadd a preparation of Rhodospirillum spp. and/or of Phaeospirillum spp.to the foodstuff during its production.

A foodstuff with cholesterol-lowering properties according to theinvention contains typically between 0.1% and 20% (w/w), preferablybetween 1% and 10% (w/w), of the food supplement according to theinvention and described above.

The present invention involves finally a preparation of Rhodospirillumspp. and/or of Phaeospirillum spp. comprising a petroleum ether extractof Rhodospirillum spp. and/or of Phaeospirillum spp. to be used in amedicament to lower the cholesterol level of blood plasma, preferably tolower the blood plasma level of LDL-cholesterol, while leaving the totalcholesterol level in blood plasma essentially unaltered and/or whileleaving the HDL-cholesterol level in blood plasma essentially unaltered.Preferably, such a preparation of the invention comprising a petroleumether extract of Rhodospirillum spp. and/or of Phaeospirillum spp.involves the species Rhodospirillum rubrum and/or the speciesPhaeospirillum molischianum.

The invention is further illustrated by the following examples, whichshould not be interpreted as limiting the present invention in any way.

EXAMPLES Experimental Procedures

The Rhodospirillum rubrum strain S1H was stored in liquid nitrogen in a10% w/w sucrose-0.85% w/w saline solution. To regrow the strain, thecells were taken out the liquid nitrogen and thawed for 30 minutes atroom temperature. Cells were streaked on a sistrom succinate agar plateand a rich Luria Bertani (LB) medium to grow colony forming units. Theagar plates were incubated at 30° C. in dark and aerobic conditions forup to 4 days.

After 4 days, 10 single colonies were picked up and transferred to 10tubes with 2 mL of Sistrom succinate liquid medium and incubated at 30°C. in dark, aerobic and orbital shaking at 150 rpm. After 4-5 days, thecells were grown and reached an OD680=0.5-0.6. To check the axenicity ofthe cultures, the cells were streaked on a Sistrom succinate agar plateand a rich LB medium agar plate and incubated up to 1 week to look forheterotrophic contaminants. When the axenicity check was approved, the 2mL cultures were transferred to 15 mL of Sistrom succinate liquid mediumand incubated at 30° C. in dark, aerobic and orbital shaking at 150 rpm.After 4-5 days the cells were grown and reached an OD680=0.5-0.6. Then,the 15 mL cultures were transferred to 100 mL of Sistrom succinateliquid medium and incubated at 30° C. in dark, aerobic and orbitalshaking at 100 rpm. After 4-5 days, the cells were grown and reached anOD680=0.5-0.6. Once the axenicity was checked on Sistrom succinate andLB medium, these cells constituted the inoculum cultures.

Cell Culturing Culture Conditions Bioreactor

Light anaerobic conditions were applied for culturing the Rhodospirillumrubrum strain S1H cells. Ten axenic inoculum cultures were pelleted bycentrifugation at 5000*g for 10 minutes. The supernatant was discardedand the pellets were pooled in 25 mL of Melissa liquid medium withacetate as carbon source (Segers & Verstraete, 1983) to constitute aconcentrated inoculum.

The bioreactor was sterilized by wet-heat sterilization, 20 minutes ofwater vapor exposure at 121° C. and 1.2 bar in an autoclave. Aftersterilization the bioreactor was closed. Bottles with Melissa medium, 1M H₂SO₄, and for the effluent were coupled aseptically in a laminar flowcabinet (LAF).

Extraction Protocols

30 g of bacterial pellet of Rhodospirillum rubrum strain S1H cells, aremixed using 440 ml of a biphasic mixture of petroleum ether (boilingpoint 60-80° C.) and methanolic saline during 2 h at room temperature(biphasic mixture: 220 ml containing 20 ml of NaCl 0.3% by mass and 200ml of methanol+220 ml of the petroleum ether (boiling point 60-80° C.)).After centrifugation at 5000 RPM during 20 minutes at room temperature,the upper phase (the petroleum ether phase) was removed and stored atroom temperature. Lower phase (the methanolic saline solution) wasoptionally submitted to a second extraction with an additional 220 ml ofpetroleum ether for 2 h at room temperature. The second upper phase wasmixed with the first one and dried using a rotavapor system. Theresulting viscous liquid was named “FRACTION I.1”, or “fI.1”, or“Extract I.1” in FIG. 1.

For obtaining an alternative extract of Rhodospirillum rubrum strain S1Hcells, 260 ml of CHCl₃/CH₃OH/NaCl 0.3% (9:10:3) solution was added tothe above mentioned lower phase (methalonic saline solution) and thenincubated under agitation during 4 h at room temperature. After removinginsoluble bacterial material by filtration, the supernatant was mixedwith 145 ml of CHCl₃ and 145 ml of NaCl 0.3% and then incubated 1 hunder agitation at room temperature. Finally, the resulting organicphase (lower phase) was removed and dried using a rotavapor and named“FRACTION I.2”, or “fI.2”, or “Extract I.2” in FIG. 1.

A third extract, “Extract III”, or “FRACTION III”, or “fIII”, wasobtained as follows. Membranes of Rhodospirillum rubrum strain S1H cellswere isolated from 30 g of bacterial pellet which was washed in 5 mMphosphate buffer, pH 7.0. Washed bacterial pellet was broken bysonication (3 times for 30 sec, 100 amplitude at 4° C.), 1 Freeze-thawcycle and 2 French Press cycle (Thermo, High pressure cell, 1500 psi)cycle. The bacterial homogenate was centrifuged at 5,000 rpm for 5 minto remove cellular debris, and the supernatant from this firstcentrifugation step was centrifuged in a second centrifugation step for30 min at 15,000 rpm to pellet the membranes. Extract III is thesupernatant after the second centrifugation step.

Materials and Methods for Mass Spectrometry Analysis MALDI-ToF

Matrix-assisted laser desorption ionization time-of-flight (MALDI-ToF)mass spectrum was recorded using a QToF Premier mass spectrometerequipped with a Nd:YAG laser, operating at 355 nm with a outputfrequency of 50 Hz. Time-of-flight mass analyses were performed inreflection mode at a resolution of about 10.000. Samples of fI.1 andfI.2 were analyzed using(DCTB)trans-2-[3-(4-tertbutylphenyl)-2-methylprop-2-enylidene]malononitrile. This matrix was prepared as a 40 mg/mL solution in CHCl₃.The matrix solution (1 mg/mL) was applied to a stainless steel targetand air dried. The samples were dissolved in THF and 1 microliteraliquots of this solution were applied onto the target area alreadybearing the matrix crystals and air dried. For the recording of thesingle-stage MS spectra, the quadrupole (rf-only mode) was set to passions from 200 to 2500 Th, and all ions were transmitted into the pusherregion of the time-of-flight analyzer where they were mass analyzed with1 s integration time.

Q-tOF (5600 ABSCIEX)-nanoESI-MS

Samples of fI.1 and fI.2 were diluted in 0.1% formic acid inacetonitrile, centrifuged at room temperature during 5 min at 13.000 RPMand the supernatants were infused directly in the Mass spectrometer(flow rate: 89 microliter/hour) using nano-esi source. The acquisitionparameters were: ion source gas1: 4; Curtain gas 15; ionspray Voltagefloating 2.300, heater temperature 150° C.; Polarity: positive; ToF massrange: 100-2.000.

Ion Trap (HCT Ultra Brucker)-nanoESI-MS

Samples of fI.1 and fI.2 were diluted in 0.1% formic acid inacetonitrile, centrifuged at room temperature during 5 minutes at 13.000RPM and the supernatants were infused directly in the Mass spectrometer(flow rate: 89 microliter/hour) using nano-esi source. The acquisitionparameters were: capillary 1.900 Volt; Dry gas: 6 l/min; Dry temp: 250°C.; Polarity: positive; scan mode: Standard-enhanced; scan range:100-2.000; Smart target 20.000; Max accu time: 200 ms.

Results

MALDI-ToF analyses of fI.1 and fI.2 showed that fI.1 and fI.2 comprise anumber of compounds. In FIG. 2 the results of the Maldi Tof analysis ofthe fraction I.1 and the fraction I.2 are shown. Ten μl of each fractionwas dried in presence of matrix.

Using Maldi-ToF data, Carotenoids and Quinones were observed in fI.1(molecular ion indicated in brackets as M+H+value): rhodovibrin (585.5),1-hydroxy-spirilloxanthin (583.5), 3,4-didehydrorhodopin (587.5),chloroxanthin (557.5), bacteriopheophytin a (BPha; 883.5), rhodopsin(555.4), spirilloxanthin (597.4), 3,4-dihydrospirilloxanthin (599.5),phytyl derivative of bacteriopheophytin a (BPha (phytyl); 889.5),ubiquinol-10 (865.7), ubiquinone-9 (795.6), ubiquinone-10 (848.7) andrhodoquinone-10 (863.7). The same carotenoids and quinones wereidentified in the fI.1 extract when applying nano-ESI Q-ToF analysis.

Maldi-ToF data analysis with the fI.1 extract revealed, amongst others,peaks at the following m/z values (approximate relative intensity inbrackets): 500.3 (1); 882.5 (1); 898.5 (4).

Maldi-ToF data analysis with the fI.2 extract revealed, amongst others,peaks at the following m/z values (approximate relative intensity inbrackets): 500.3 (0); 882.5 (3); 898.5 (1).

Analysis of the fI.1 extract using Q-tOF (5600 ABSCIEX)-nanoESI-MSconfirmed the presence of the above listed Carotenoids and Quinonespresent in fI.1. The NanoESI MS spectra are obtained for the fractionI.1 and the fraction I.2 with a triple tof mass spectrometer (ABSCIEX)using acetonitrile 99%, 1% HCOOC as organic solvent.

In the Q-tOF (5600 ABSCIEX)-nanoESI-MS spectrum for fI.1 amongst others,peaks were revealed at the following m/z values (approximate relativeintensity in brackets): 647.6 (2.5); 651.6 (3.3); 881.5 (2); 927.5 (1).

In the Q-tOF (5600 ABSCIEX)-nanoESI-MS spectrum for fI.2 amongst others,peaks were revealed at the following m/z values (approximate relativeintensity in brackets): 647.6 (2-3); 651.6 (16); 881.5 (5); 927.5 (1).

In the Ion Trap (HCT Ultra Brucker)-nanoESI-MS spectrum for fI.1 amongstothers, peaks with relative high intensities were revealed at thefollowing m/z values 652.3; 686.4; 690.6; 752.7; 769.3; 881.9; 897.8;913.8; 927.7. NanoESI MS spectra are obtained for the fraction I.1 andthe fraction I.2 with an IonTrap mass spectrometer (Bruker) using 1%HCOOC in acetonitrile.

The petroleum ether extract fI.1 of Rhodospirillum rubrum comprisedcompounds with a molecular weight of at least 2100 Da giving rise topeaks having an m/z value of 752.7 and 769.3 in a nanoESI-MS spectrumobtainable with the IonTrap nanoESI-MS high-resolution tripletime-of-flight mass spectrometer (Bruker), said peaks with an m/z valueof 752.7 and 769.3 having an intensity in the nanoESI-MS spectrum thatis about five times the intensity of two further peaks in the nanoESI-MSspectrum having an m/z value of 665.8 and 883.6.

In the Ion Trap (HCT Ultra Brucker)-nanoESI-MS spectrum for fI.2 amongstothers, peaks with relative high intensities were revealed at thefollowing m/z values 665.8; 883.6; 899.8; 915.6.

The petroleum ether extract fI.2 of Rhodospirillum rubrum comprisedcompounds giving rise to peaks having an m/z value of 665.8 and 883.6 ina nanoESI-MS spectrum obtainable with the IonTrap nanoESI-MShigh-resolution triple time-of-flight mass spectrometer (Bruker), saidpeaks with an m/z value of 665.8 and 883.6 having an intensity in thenanoESI-MS spectrum that is at least five times the intensity of twofurther peaks in the nanoESI-MS spectrum having an m/z value of 752.7and 769.3.

Mice Test—Effect of fI.1, fI.2 and fIII on Plasma Cholesterol Level

The mice test for testing the influence of a diet comprising fI.1 orfI.2 or fIII on plasma cholesterol level was performed at SCK•CENanimalarium (BE) following 2 weeks acclimation of 40 C57BL/6 male mice.After initial weighing of the food and the mice, they were placed inindividual ventilated cage. Food consumption was checked every day andhydrogel weighed every 2 days. Based on previously performed preliminarypalatability tests, the bacterial extracts were resuspended in sunfloweroil, and 5% regular sugar was added to the chow (Cafetaria-diet) toensure high palatability.

The first week, the first group of mice received the CafetariaDiet+sunflower oil replacing R. rubrum extract fI.1 or fI.2 or fIII adlibitum while the second group of mice received the Cafetaria Diet+thecontrol diet replacing R. rubrum extract fI.1 or fI.2 or fill adlibitum.

The second week, the control group continued on the same diet while thethree experimental groups received the Cafetaria Diet+10% of either thefI.1 extract or the fI.2 extract or the fill extract in sunflower oil.Thus, one group of mice was fed with feed comprising the fI.1 petroleumether extract of Rhodospirillum rubrum, a second group of mice was fedwith feed comprising the fI.2 extract of Rhodospirillum rubrum, a thirdgroup of mice was fed with feed comprising the fIII extract ofRhodospirillum rubrum, a fourth group of mice, the control group, wasfed feed without any of the extracts of Rhodospirillum rubrum.

Effects of feeding control feed or feed comprising either fI.1 or fI.2or fill on cholesterol levels in plasma is detailed below.

End of Mice Test

After 2 weeks of testing, the mice where weighted and euthanized usingintraperitoneal pentobarbital injection prior to dissection. Whole-bloodwas removed in EDTA-tubes, centrifuged to obtain plasma and placed at 4°C. for further analysis.

Mice Weight

After week 1 and week 2, no differences between the groups weredetected.

Blood Analysis Total Cholesterol, HDL and LDL Fractions

FIG. 1 shows the results of the cholesterol-, HDL- and LDL analysis.

Extract I.1 (which is Fraction I.1, fI.1) has a significant effect onthe plasma LDL-cholesterol fraction since it decreased it for more than40%, i.e. about 46%, compared to the control group (p<0.001) while thetotal cholesterol stayed essentially unchanged. See FIG. 1. Furthermore,also the HDL-cholesterol level in the mice group that were fed the fI.1fraction, stayed essentially unaltered after the experimental period.Total cholesterol in plasma was 4.39 μg/μl for the fI.1 group of micecompared to 4.46 μg/μl for the control group, HDL-cholesterol was 2.93μg/μl for the fI.1 group and 2.72 μg/μl for the control group, andLDL-cholesterol was 0.36 μg/μl for the fI.1 group (p<0.001) and 0.67μg/μl for the control group, respectively.

Extract I.2 (which is Fraction I.1, fI.2), however, did not have asignificant effect on any of the cholesterol levels when totalcholesterol, HDL-cholesterol and LDL-cholesterol levels were concerned.See FIG. 1. Total cholesterol in plasma was 4.45 μg/μl for the fI.2group of mice compared to 4.46 μg/μl for the control group,HDL-cholesterol was 3.00 μg/μl for the fI.2 group and 2.72 μg/μl for thecontrol group, and LDL-cholesterol was 0.60 μg/μl for the fI.2 group(p<0.001) and 0.67 μg/μl for the control group, respectively.

Extract III (which is Fraction III, fIII), similar to Extract I.1, alsodid not have a significant effect on of the cholesterol levels whentotal cholesterol, HDL-cholesterol and LDL-cholesterol levels wereconcerned. That is to say, total cholesterol in plasma remainedunaltered at 4.46 μg/μl for the fill group of mice compared to thecontrol group, HDL-cholesterol was 2.93 μg/μl for the fill group and2.72 μg/μl for the control group, and LDL-cholesterol was 0.60 μg/μl forthe fill group and 0.67 μg/μl for the control group, respectively.

From this mice test using R. rubrum S1H extracts fI.1 and fI.2 and fIII,it is obvious that the petroleum ether extract fI.1 has a beneficialeffect on lowering LDL-cholesterol level to a large extent, while at thesame time keeping the HDL-cholesterol level essentially unaltered whenthe extract is administered orally. The mice test using R. rubrum S1Hextract fI.2 or extract fill revealed that the compounds in theseextracts do not significantly influence total cholesterol,HDL-cholesterol and LDL-cholesterol levels when the extracts areadministered orally.

REFERENCES

-   D. E. Minnikin, A. G. O'Donnell, M. Goodfellow, G. Alderson, M.    Athalye, A. Schaal and J. H. Parlett, “An integrated procedure for    the extraction of bacterial isoprenoid quinones and polar lipids”,    Journal of Microbiological Methods 2 (1984) pp. 233-241 (Elsevier).-   Segers, L. and Verstraete, W., 1983, Conversion of organic acids to    H₂ by Rhodospirillaceae grown with glutamate or dinitrogen as    nitrogen source. Biotechnol. Bioeng. 1983; 25: 2843-2853.-   William W. Parson and Harry Rudney, “The Biosynthesis of Ubiquinone    and Rhodoquinone from p-Hydroxybenzoate and n-Hydroxybenzaldehyde in    Rhodospirillum rubrum”, The Journal of Biological Chemistry, Vol.    240, No. 4, April 1965, pp. 1855-1863.

1. An extract of Rhodospirillum rubrum, comprising rhodovibrin,1-hydroxyspirilloxanthin, 3,4-didehydrorhodopin, chloroxanthin,bacteriopheophytin a, rhodopin, spirilloxanthin,3,4-dihydrospirilloxanthin, phytyl derivative of bacteriopheophytin a,ubiquinol-10, ubiquinone-9, ubiquinone-10 and rhodoquinone-10.
 2. Thepetroleum ether extract of Rhodospirillum rubrum according to claim 1,for use as a medicament.
 3. The petroleum ether extract ofRhodospirillum rubrum according to claim 1, for use in the lowering ofLDL-cholesterol in blood plasma of a subject.
 4. The petroleum etherextract of Rhodospirillum rubrum according to claim 1, for use in amethod for the lowering of LDL-cholesterol in blood plasma of a subject.5. The petroleum ether extract of Rhodospirillum rubrum according toclaim 1, for use in the lowering of LDL-cholesterol in blood plasma of asubject or for use in a method for the lowering of LDL-cholesterol inblood plasma of a subject, wherein the subject suffers from or has anincreased risk for any one or more of cardiovascular disease,atherosclerosis, dyslipidemia, arteriosclerosis, hypercholesterolemia,familial hypercholesterolemia, hyperlipidemia, an LDL plasma level of atleast 70 mg/dL, a total cholesterol level of at least 200 mg/dL, anLp(a) level of at least 14 mg/dL, inflammation, inflammatory disease,ischemic, or infection.
 6. The petroleum ether extract of Rhodospirillumrubrum according to claim 1, wherein the extract is administered orally.7. A food supplement with LDL-cholesterol lowering properties,comprising the petroleum ether extract of Rhodospirillum rubrumaccording to claim
 1. 8. A foodstuff comprising the food supplementaccording to claim
 7. 9. A method for the production of the petroleumether extract of Rhodospirillum rubrum of claim 1, comprising the stepsof: a) providing Rhodospirillum rubrum cells b) providing a biphasicmixture having about a 1:1 ratio by volume of petroleum ether that has aboiling point between 60° C. and 80° C. and methanol that comprisesabout 0.03% by weight of sodium chloride; c) mixing the cells of step a)with the biphasic mixture of step b) for about 1 to 3 hours at atemperature between 15° C. and 25° C.; d) separating the petroleum etherphase from the methanol phase of the biphasic mixture; and e) isolatingthe petroleum ether phase, thereby obtaining the petroleum ether extractof Rhodospirillum rubrum.
 10. A method of treating a subject in needthereof, the method comprising administering an extract ofRhodospirillum rubrum, comprising rhodovibrin, 1-hydroxyspirilloxanthin,3,4-didehydrorhodopin, chloroxanthin, bacteriopheophytin a, rhodopin,spirilloxanthin, 3,4-dihydrospirilloxanthin, phytyl derivative ofbacteriopheophytin a, ubiquinol-10, ubiquinone-9, ubiquinone-10 andrhodoquinone-10.
 11. The method of claim 10, wherein the method lowersLDL-cholesterol in blood plasma of the subject.
 12. The method of claim10, wherein the subject suffers from or has an increased risk for one ormore of cardiovascular disease, atherosclerosis, dyslipidemia,arteriosclerosis, hypercholesterolemia, familial hypercholesterolemia,hyperlipidemia, an LDL plasma level of at least 70 mg/dL, a totalcholesterol level of at least 200 mg/dL, an Lp(a) level of at least 14mg/dL, inflammation, inflammatory disease, ischemia, or infection. 13.The method of claim 10, wherein the method comprises administering theextract orally.